To date, in vivo molecular imaging agents specifically target the cell surface or microenvironment. However, most of the highly specific changes of cancer cells that differentiate cancers cells from normal cells occur intracellularly. The challenge, therefore, is to develop agents that report intracytoplasmic changes yet still are capable of being imaged in vivo. The first step in achieving this goal is to target the imaging agent to the cell surface which requires affinity for a cell surface marker. The ligand must then be internalized by endocytosis and then bind to the appropriate site whereupon it activates. These requirements place large demands on synthetic chemistry since the molecular construct must have multiple functionalities. We are developing smart activatable optical constructs which only fluoresce when they are internalized to the cytoplasm. Using a series of commercially available dyes that are bound to targeting compounds and then modified to fluoresce under specific intracellular conditions such as lower pH and in the presence of specific enzymatic activity we are making progress toward the goal of intracellular in vivo imaging. This work is being performed in collaboration with Prof. Urano from the University of Tokyo Chemistry Department. Over the past year we have made considerable advances in this area by proving that it is possible to create highly activatable optical imaging agents based on the BODIPY and Rhodamine backbones. We are also pioneering efforts to create multimodal imaging agents; agents that can be seen on both optical cameras as well as PET, MR or radionuclide cameras. The agents being designed are highly biocompatible and elements have already been used in humans. For intance, the agent Galactosylserum Albumin (GSA) which we have labeled with Rhodamine Green (GSA-RhG) is internalized rapidly within cancer cells and may be viable as an agent for human use. We are developing activated fluorescent molecular imaging agents and have a number of successes over the year. However, we continue to pursue a solution that will activate only within cancer cells and not within other, normal cells. The ability to image multiple targets simulanteously led us to explore multiexcitation and multiemission cameras. We had hoped that a single excitation light would be able to activate multiple fluorophores at differing wavelengths but this proved to be unrealistic. Instead, we use multiple wavelength excitation light using a new Maestro camera. This has allowed us to simultaneously image up to 4 targets in the near infrared and is very promising for clinical application